Nuclear hormone receptors play key roles in homeostasis and energy metabolism through their action, as gene-specific transcription factors, in metabolic tissues. Their function on specific target genes is highly dependent upon interactions with cofactors (notably the 30-subunit Mediator complex) that interface directly with the general transcription machinery and with cofactors that act indirectly to effect histone modifications (epigenetic marks) of the chromatin template. These cofactors add an important level of gene regulation, and this proposal seeks to detail biochemical mechanisms by which nuclear receptors (including PPAR?, TR and ERR) and key interacting cofactors regulate genes important for white adipocyte differentiation and function (fat storage), brown fat differentiation and function (energy dissipation through adaptive thermogenesis) and muscle function. The cofactors of special interest include the primary receptor-interacting subunit of th Mediator (MED1), factors that may provide alternative or redundant pathways for Mediator recruitment, the brown fat differentiation factor PRDM16, the inducible PGC-1 that is important for thermogenesis in brown fat, corepressors (such as RIP140) that necessitate opposing coactivator functions, histone modifying factors such as the activating p300 acetyl- and SET1/MLL methyl-transferases, and other DNA-binding regulatory factors (C/EBPs) that act synergistically with PPAR?. The mechanism of action and physiological functions of these factors on key target genes will be studied by several complementary approaches. First, we will use cell-free systems reconstituted with purified factors and DNA templates to detail mechanisms of cofactors that facilitate direct activation or repression of the general transcriptio machinery, with special emphasis on Mediator recruitment by MED1 versus other cofactors. Second, we will use cell-free systems reconstituted with purified factors and chromatin templates to detail (i) mechanisms of cofactors that directly or indirectly (as bridging proteins) effect covalent histone modifications and (ii) functions of these modifications through recognition by other effectors. Third, we will investigate the in vivo gene/tissue-specific functions of nuclea receptor coactivators during adipogenesis and adaptive thermogenesis through the generation and analysis of conditional knockout and mutant knockin mice, with emphasis on the MED1 subunit that is conditionally required for high level nuclear receptor function and whose mutation results in mice with improved glucose tolerance and insulin sensitivity as well as resistance to diet induced obesity. Fourth, through further mouse genetic and in vitro assays, we will investigate the molecular basis for the dramatic, metabolically favorable phenotype (induction of the thermogenic UCP1 and slow-twitch Type I myofiber genes; increased insulin sensitivity/glucose tolerance and resistance to diet-induced obesity) in skeletal muscle-specific Med1 knockout mice. By identification of new factors and mechanisms, and thus of novel therapeutic targets, these studies will have important implications for the control of obesity and muscle dystrophy. PUBLIC HEALTH RELEVANCE: Obesity and Type II diabetes represent worldwide health problems and result from imbalances in homeostasis and energy metabolism in metabolic tissues such as fat and muscle. The present proposal seeks to understand the molecular basis for the regulation, by nuclear hormone receptors and associated cofactors, of genes that regulate the differentiation and function of white fat (involved in energy storage), brown fat (involved in energy dissipation and newly recognized as important metabolic tissue in adult humans) and muscle. The results will have important implications for possible therapeutic approaches to these and other health problems.